Project Summary Muscle-invasive bladder cancers (MIBCs) represent an aggressive subset of bladder tumors that are associated with high mortality rates despite intensive multimodality treatment. DNA-damaging agents like cisplatin play a key role in the treatment of MIBC and many other solid tumors, yet validated biomarkers of response to DNA-damaging therapy are lacking. Recently, an association between somatic mutations in ERCC2, a core member of the nucleotide excision repair (NER) pathway, and improved response to cisplatin-based chemotherapy was uncovered in MIBC, representing one of the first validated examples of an association between a tumor DNA repair pathway alteration and response to a DNA-damaging agent. Preliminary functional analysis suggests that the observed ERCC2 mutations result in loss of NER capacity; however, the functional underpinnings of this association across tumors and clinical contexts are not known. This proposal aims to characterize the role of mutations in ERCC2 and other DNA repair genes in MIBC biology and treatment response. One of the limitations to understanding the biological relevance of novel DNA repair alterations in tumors is the lack of robust, efficient assays to test the functional effects of observed mutations. The first aim of this application will employ a novel high-throughout, fluorescence-based microscopy assay to measure the ability of ERCC2 mutations to support cellular NER. The assay will be applied to all ERCC2 mutations observed across several MIBC cohorts, and findings will be interpreted in the context of available treatment response and patient outcome data in order to define the functional landscape of ERCC2 mutations and develop a framework for predicting functional effects and therapeutic implications of ERCC2 mutations. Although MIBCs harboring ERCC2 mutations have improved response to cisplatin-based chemotherapy, the mechanism by which heterozygous ERCC2 mutations confer sensitivity is not known. The second aim will utilize a combination of cellular and biochemical approaches to dissect the effect of mutations on ERCC2 protein function, cellular properties, and sensitivity to established and emerging MIBC therapies. In addition, the effect of ERCC2 mutations on tumorigenesis will be investigated by introducing ERCC2 mutations into a normal human urothelial cell line alone and in combination with other known MIBC driver mutations. The third aim will investigate the hypothesis that mutations in ERCC2 (or other DNA repair genes) define a subset of MIBC patients who are ideal candidates for bladder-preserving treatment with concurrent cisplatin-based chemoradiotherapy (CRT). Targeted sequencing of ERCC2 and 1000 additional cancer genes will be performed in two large cohorts of MIBC patients treated using CRT. These analyses are likely to further define a role for ERCC2 as a biomarker in MIBC and may have broad implications for understanding the role of DNA repair pathway alterations in a variety of tumor settings.